Abstract
Harnessing the biology of regulatory T cells (Tregs) for therapeutic development is one of medicine’s most promising opportunities to transform disease treatment. Initially viewed simply as guardians against destructive immune responses, we now understand that Tregs are adaptive and highly specialized coordinators of immune tolerance and tissue repair. This strategic roadmap examines how evolving insights into their central role in maintaining tolerance and health can transform therapeutic development across medical specialties. Early efforts to evaluate Treg therapies have proven safe and shown some clinical benefit. The convergence of biological insights and technological advances has the potential to harness and exploit this specialized tolerogenic population by augmenting function through environmental cues and reinforcing tissue-repair capabilities. Advanced engineering approaches can now endow Treg therapies with antigen-specificity, enforce suppressive programming, and enable off-the-shelf or in vivo gene therapy opportunities. This new class of therapeutics promises to shift treatment paradigms from lifelong management to drug-free durable remissions or even cures for diseases currently requiring chronic immunosuppression. The recognition that dysregulated inflammation underlies countless human diseases opens unprecedented possibilities for treating intractable conditions across specialties—from transplantation and autoimmunity, metabolic disorders and cardiovascular disease, to neurodegeneration and even aging itself. Realizing the potential of Treg-based therapies remains a primary goal for the field, representing a strategic shift from systemic immunosuppression to precision tolerance restoration as a unifying therapeutic paradigm.
Key points
Regulatory T cells (Tregs) are adaptive, tissue-specialized regulators that maintain immune tolerance and tissue homeostasis, rather than acting only as suppressor cells.
Dysregulated inflammation underlies many diseases, positioning Treg-based tolerance restoration as a therapeutic strategy beyond traditional transplant and autoimmune indications.
Early clinical studies have established a foundation of safety and feasibility, while next-generation approaches aim to improve specificity, persistence, stability, and scalability.
Precision tolerance restoration depends on matching the Treg strategy to disease biology, tissue context, and timing of intervention.
The field is moving beyond early polyclonal Treg therapies toward more targeted approaches, including antigen-specific, engineered, and potentially off-the-shelf or in vivo strategies, designed to improve precision, persistence, and scalability.
Introduction
The human immune system faces a formidable challenge: distinguishing harmful pathogens from harmless or beneficial agents. Its task is to maintain a precarious balance: mounting responses against threats while actively preserving tolerance toward harmless agents. Since the seminal identification of the regulatory pathways that govern this balance (Figure 1), immunity has been redefined as a system governed not only by activation but also by active regulation. Regulatory T cells (Tregs), a specialized class of immune cells described by the Nobel Committee as the immune system’s “security guards,” () prevent other immune cells from attacking the body’s own tissues and maintain peripheral immune tolerance. The transcription factor forkhead box P3 (FOXP3) is the key genetic regulator of these cells, controlling their development and function and enabling them to suppress harmful autoimmune responses.
Figure 1
Traditionally seen as a defense system against pathogens, toxins, and xenobiotics, the immune system is equally one of tolerance: it must prevent destructive self-reactivity and inappropriate responses to environmental antigens, limit maladaptive aftermaths of immune responses, adapt to injury and other stresses, and maintain peaceful coexistence with commensal microbes (). This dual mandate—defense versus tolerance—constitutes the essential functions of the immune system (Figure 2). At the center of this tolerance system lie mechanisms that actively restrain unwanted responses and preserve the fragile homeostasis that is vital for human health.
Figure 2
Beyond the “usual suspects” of autoimmunity and transplant rejection, dysregulated tolerance underlies a remarkably broad spectrum of pathologies, including cancer immune evasion (), hyperinflammatory responses to infection such as severe COVID-19 (), neurodegeneration (), metabolic disease (), fibrosis (), pregnancy/reproductive diseases (), and aging ()—each driven by overlapping yet distinct inflammatory processes. Current therapies typically rely on broad immunosuppression, targeting end-stage inflammation that arises long after substantial dysregulation and tissue damage have occurred.
The next frontier is “precision tolerance restoration,” a multidimensional framework that integrates three distinct axes of specificity to transcend non-specific suppression. First is product precision: a reference to the biological design of the therapeutic agent, including antigen specificity, stability programming, metabolic fitness, and resistance to inflammatory conversion. Second is delivery precision, which reflects the capacity to localize regulatory activity to relevant tissues through trafficking receptors, engineered targeting domains, or controlled persistence. Finally, precision in patient selection involves stratification by immunological endotype, disease stage, or predictive biomarkers.
Current clinical evidence most strongly supports advances in product and delivery precision, while systematic biomarker-guided patient stratification is still evolving. Importantly, the requirement for precision varies by context. In transplantation and some autoimmune settings, polyclonal Tregs may effectively exploit bystander suppression (the ability to suppress immune responses to neighboring antigens) and infectious tolerance (the propagation of a regulatory state to other immune cells) (), whereas defined autoantigen contexts may benefit from antigen-targeted approaches. Thus, precision is not a monolithic requirement but a context-dependent continuum; the degree of engineering required is dictated by the specific immunological environment and the underlying mechanism of dysregulation. However, there are multiple pre-clinical examples showing a clear benefit in engineering Tregs to express antigen-specific receptors ().
Immune tolerance is established through two fundamental, non-redundant pathways. Central tolerance eliminates self-reactive T and B cells during education in the thymus and bone marrow, respectively (, ). Peripheral tolerance acts as a second line of defense, restraining autoreactive cells that escape central deletion () through cell-intrinsic and cell-extrinsic mechanisms. Cell-intrinsic tolerance limits activation and effector function within individual immune cells through mechanisms such as T-cell anergy and exhaustion (, ). Cell-extrinsic tolerance relies on specialized immunomodulatory cells that act dominantly in trans. Among these, Tregs serve as master regulators of immunity, coordinating peripheral tolerance across tissues (). Together, these pathways maintain immune equilibrium, and their disruption underlies diverse immune pathologies (). Both intrinsic and extrinsic tolerance mechanisms are now recognized as therapeutic targets. For example, inhibitory receptor signaling pathways provide adjustable cell-intrinsic brakes on immune activation, and their blockade has transformed cancer treatment by enabling tumor recognition and destruction (). Conversely, Tregs mediate potent cell-extrinsic suppression, making them uniquely suited for therapeutic restoration of immune balance.
The discovery of Tregs, recently recognized by the Nobel Foundation for its profound impact on human health (), redefined immunity as a system governed not only by activation but also by active regulation. Tregs are remarkably versatile, controlling immunity through mechanisms far beyond their classical suppressor role. They orchestrate tissue repair () and metabolic balance (), making them uniquely adaptable therapeutic agents: living drugs that act not as blunt, non-specific suppressors but as architects of immune homeostasis.
From an evolutionary perspective, Tregs endowed the immune system with a mechanism to balance offense with restraint, enabling a powerful yet sustainable immunity—an adaptation essential for long-lived, complex organisms (). However, this same potency creates a therapeutic duality: the same circuits that protect against autoimmunity and graft rejection can, when unchecked, blunt antitumor and antiviral responses (). This duality makes Tregs a focal point for therapies that expand or stabilize endogenous populations or mimic their effects through parallel regulatory pathways versus eliminating the cells, to unleash immunity in settings such as cancer ().
The field of Treg medicine now stands at a critical inflection point, propelled by converging insights into Treg biology, rapid technological innovation, and accelerating clinical translation. Early clinical trials have shown that Treg therapies are safe and exhibit early signs of efficacy in transplantation and autoimmune disease, underscoring their broader potential () (Figure 3). Yet this is only the end of the beginning. Emerging tools, from genetic engineering to rational drug combinations, are poised to transform how tolerance is induced and maintained. This new era of medicine will be guided by two central questions: what transformational Treg capabilities can be unlocked in patients and how can they be harnessed therapeutically? This article addresses these questions by highlighting recent breakthroughs in Treg biology with emerging therapeutic technologies to establish a strategic roadmap for the era of precision tolerance medicine.
Figure 3
Advances in Treg cell biology: the spatiotemporal tolerance program
Tolerance as adaptation
Tregs are best understood as nature’s polypharmacy—living drugs that deploy multiple synergistic mechanisms to maintain durable tolerance (). The traditional view of Tregs as simple immunologic brakes understates their true nature. These cells act as conductors, coordinating diverse mechanisms and cell types to form dynamic, self-reinforcing tolerance networks that function as a complex adaptive system (Figure 4). This helps explain why therapies that flip a single switch so often fail: tolerance is not governed by one lever but by an integrated set of switches and dials operated in concert. In this sense, tolerance functions less as a passive brake and more as an actively programmed system directed by Tregs to maintain stability and limit tissue damage.
Figure 4
Individual Tregs deploy complementary suppressive tools that build resilience within the tolerance network. They secrete tolerogenic cytokines such as interleukin 10 (IL-10) () and transforming growth factor β (TGF-β) (, ) and engage inducible inhibitory pathways, including cytotoxic T lymphocyte-associated protein 4 (CTLA-4) () and T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) (), to restrain activation across immune populations. Metabolic control adds another layer: Tregs induce indoleamine 2,3-dioxygenase (IDO) to deplete tryptophan and generate immunosuppressive kynurenines (). By efficiently consuming IL-2 through high-affinity IL-2 receptors (CD25), Tregs stabilize their lineage while depriving effector T cells (Teff) of this critical growth factor, creating a producer–consumer circuit that converts pathogenic activation into regulatory fuel (). They also express CD39 and CD73, which convert extracellular adenosine triphosphate and adenosine diphosphate into anti-inflammatory adenosine, providing a complementary metabolic route of suppression (). In parallel, contact-dependent mechanisms reinforce suppression: trogocytosis removes costimulatory molecules from antigen-presenting cells (APCs) (, ), and Tregs can release granzymes to eliminate target cells (), though this pathway is context-dependent (). When one pathway is constrained, others compensate, underscoring the fail-safe, redundant design of tolerance as a resilient system.
These individual mechanisms culminate in a broader function: the establishment of non-inflammatory, tissue-supportive microenvironments that extend beyond direct Treg targets (). Cytokine secretion and metabolic modulation remodel local surroundings, generating localized fields of tolerance. Studies using Tregs expressing a myelin basic protein (MBP)-specific T-cell receptor (TCR) illustrate this principle: though engineered to target MBP, these cells suppressed T cells with different antigen specificities in multiple sclerosis (MS) models. This contact-independent bystander suppression required IL-2 produced by Teff, showing how Tregs co-opt pathogenic activation signals to sustain suppression (). Similar principles have been observed in type 1 diabetes (T1D) (, ), hemophilia A (), and anti-glomerular basement membrane disease (). The therapeutic implication is striking. Rather than matching every inflammatory antigen, success may be achieved by delivering a single, well-targeted Treg population to the diseased site, where it can establish suppressive microenvironments that restrain diverse effector cells. This ability to convert narrow specificity into broad tolerance exemplifies how Tregs operate as an adaptive, tunable network, forming broad but localized feedback loops with effector cells.
While bystander suppression offers immediate spatial reach, the long-term stability of the tolerance network relies on infectious tolerance—the capacity to propagate the regulatory state over time (). This creates self-sustaining circuits that amplify suppression long after therapeutic cells are gone (44, 45). Recent studies with human leukocyte antigen (HLA)-A2-specific chimeric antigen receptor (CAR)-Tregs illustrate this vividly. In mice receiving an HLA-A2+ islet transplant, donor-specific CAR-Tregs not only induced tolerance to HLA-A2 but also conferred cross-protection, preventing islet-specific T cells from driving autoimmune diabetes. Remarkably, protection persisted even after therapeutic CAR-Tregs were removed, marked by the emergence of islet antigen-specific CD4+FOXP3+ cells, providing direct proof of infectious tolerance (46).
Tregs also collaborate with stromal and myeloid partners to promote tissue-level tolerance. TGF-β (47) and IL-10 (48) condition APCs, reducing their capacity to elicit proinflammatory T-cell responses while promoting new regulatory populations. Factors such as IL-33 (49), tumor necrosis factor receptor 2 (TNFR2) signaling (50–52), and tissue-repair programs (53) establish positive feedback loops that stabilize tolerance networks. Stromal cells are pivotal in this process: fibroblasts (54), endothelial cells (55–57), epithelial cells (58), and mesenchymal stromal cells (59, 60) provide addressins, chemokines, and metabolites that recruit and potentiate Tregs. In turn, Tregs reprogram stroma toward reparative and tolerogenic states (61–63). This crosstalk anchors regulatory networks within local tissues, ensuring that, once established, tolerance becomes durable and self-reinforcing.
Collectively, these findings demonstrate that tolerance is not merely “remembered” but actively maintained and propagated, establishing a stable equilibrium that persists long after the initiating signal fades. This network-building capacity positions Tregs as central nodes in an adaptable tolerance system, far more dynamic than the traditional “immunologic brake” view. The clear path forward is to optimize therapeutic Tregs to strengthen and propagate existing regulatory circuits, transforming single or limited antigen-specific therapies into durable, tissue-wide immune regulators that amplify the effects of the infused cells. Disease, in this framework, represents a breakdown in redundancy that therapeutic intervention must reverse by rebuilding these self-sustaining loops.
These capabilities position Tregs as a prototype for precision cell-based medicine, offering a new therapeutic paradigm that programs rather than simply suppresses immune responses. The opportunity ahead lies in harnessing the innate capacity of Tregs to stabilize and extend local tolerance networks beyond their initial targets. Strategies that optimize homing, persistence, and tissue communication may ultimately prove more effective than those focused solely on maximal suppression. In short, tolerance works because it is networked, redundant, and self-amplifying: a living system that can be programmed to achieve durable disease control.
Realizing the full therapeutic potential of Tregs will require new ways to measure and define success. Unlike conventional drugs with a single target and readout, Tregs act through interconnected, adaptive mechanisms that reshape the immune ecosystem. This complexity requires rethinking how we evaluate, manufacture, and regulate living medicines. Emerging tools such as Treg molecular fingerprinting—defining shared molecular and phenotypic profile that ensures key functions are preserved across patients and sources (64–66)—may enable more consistent and predictive therapies. Realizing this vision will require close collaboration between discovery and manufacturing teams to align product specifications with biological efficacy. Moving beyond traditional drug development pipelines and single endpoints toward systems-level frameworks that reflect Tregs’ adaptive, polypharmaceutical nature will accelerate progress. Ultimately, advancing tolerance-based therapies will depend on viewing tolerance itself as a living, adaptive system.
Division of labor: from redundancy to specialization
For decades, Tregs were seen as a single, monolithic population, deployed wherever tolerance was needed. That view has shifted: Tregs are now recognized as a diverse, adaptive lineage following a spatiotemporal tolerance program, shaped by where and when they acquire their regulatory identity (67). They arise not only in the thymus (Tregs), programmed for self-recognition, but can also be induced from naive T cells in the periphery (pTregs) in response to cues such as TGF-β (68). These distinct origins generate subsets finely tuned to each tissue’s antigenic and environmental context. Once established, Tregs continue to adapt, acquiring traits that mirror the surrounding immune and tissue milieu (69). For example, Tregs at sites of T helper (Th)1 activity adopt Th1-like traits that enable them to modulate cell-mediated inflammation (70–72), while those in Th2 environments promote repair and allergic regulation (73–75). Tissue specialization extends further: skin-resident Tregs marked by GATA3 expression couple immune regulation with barrier maintenance and repair (76), whereas adipose-resident Tregs expressing peroxisome proliferator-activated receptor-γ (PPARγ) integrate immune control with metabolic regulation. In obese mice, PPARγ+ Tregs enhance insulin sensitivity, highlighting their potential as therapeutic targets for metabolic inflammation in obesity and type 2 diabetes (77). While these studies highlight organ-specific adaptations, recent systematic mapping suggests that most tissue-resident pools of Tregs are not composed of fixed populations. Instead, tissue Tregs largely share common phenotypes and TCR sequences, maintaining a “pan-tissue” surveillance program through transient residency and multi-tissue migration (78). This suggests that, while the “pan-tissue” program provides a baseline of surveillance, specialization emerges as a dynamic, site-specific response to the local environment.
The gut provides a clear example of tissue-specialized Tregs, as the local immune system must distinguish commensal microbes and dietary antigens from genuine threats. Here, a subset of pTregs expressing retinoic acid-related orphan receptor γt (RORγt+) navigates this antigenically complex environment (79). Unlike tTregs, RORγt+ pTregs are programmed for the intestinal niche, where integrin β8 (ITGB8)-mediated activation of TGF-β drives their differentiation (80, 81). Short-chain fatty acids (SCFAs) (82, 83), secondary bile acids (84, 85), and other gut-derived metabolites further refine their function through epigenetic modification of the FOXP3 locus. Together, these cues imprint a local program of tolerance that tTregs cannot replace. The disruption of gut pTregs leads to the expansion of Th2 and Th17 cells and colitis, even when other Tregs remain intact, underscoring their unique role in maintaining tolerance beyond the self (86). RORγt+ pTregs are also resilient to inflammatory stress, maintaining IL-10 production and suppressive capacity under conditions that destabilize other subsets (87, 88). This safeguard allows them to preserve function in highly inflammatory environments—a design principle that could guide next-generation Treg therapies. Moreover, mechanistic studies in mice have shown that microbiota-dependent gut pTregs can influence distal tissues such as injured muscle, demonstrating their capacity to disseminate tolerance beyond the intestine (89). This mobility, along with broad ligand recognition, shows that tolerance operates as a distributed, spatiotemporal system rather than a fixed local state.
Treg specialization also extends to the central nervous system (CNS), an immune-privileged environment that recruits these cells in response to injury or disease. Within the CNS, Tregs secrete IL-10 to limit inflammation and amphiregulin (AREG) to promote neural tissue repair, demonstrating that the spatiotemporal code of tolerance operates even in immune-shielded tissues (90, 91). Harnessing CNS Tregs could enable precision approaches for MS (92), neurodegeneration (93), and stroke (94). Overall, each specialized Treg subset contributes distinct functions that collectively define the architecture of tolerance and provide blueprints for next-generation therapies.
Most current Treg therapies rely on circulating, thymus-derived cells that are relatively unprogrammed and lack the adaptations of tissue-resident counterparts. Yet Tregs are inherently plastic, capable of adapting once they enter tissues. In many sites, pTregs acquire homing receptors that dictate localization and function. For example, gut-derived pTregs express integrin α4β7, which binds mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on endothelial cells of mucosal lymphoid tissues and the intestinal lamina propria (95). Analogous receptor–ligand pairs guide other tissue-specialized Tregs to the sites where they are best suited to act (96–98). Chemokine receptors such as C-X-C motif chemokine receptor 3 (CXCR3) (99), C-C chemokine receptor (CCR)5 (100), and CCR4 (101) also direct tTregs to inflamed tissues, indicating that both specialized and general homing mechanisms shape Treg distribution (102). Understanding these pathways may provide a roadmap for next-generation Treg therapies, enabling Tregs cells that are preprogrammed to selectively target disease-relevant tissues and re-establish tolerance.
Future therapeutic strategies will leverage the intrinsic advantages of tissue-specialized Tregs. Rather than relying on circulating, unprogrammed cells to localize passively through inflammatory cues, engineered or selected Tregs endowed with defined tissue-homing and metabolic properties could migrate precisely and function durably at target sites. Although tissue-resident Tregs are rare and difficult to isolate, their features can be reproduced through gene-editing approaches that encode key homing receptors, transcriptional programs, and metabolic adaptations (103). Embedding this spatiotemporal code of tolerance into therapeutic design could yield treatments with enhanced precision, delivering targeted immune regulation while reducing off-target immunosuppression.
Tregs as architects of repair
Beyond their well-established role in maintaining tolerance, Tregs have emerged as architects of tissue repair, expanding their therapeutic potential into dual-function platforms that couple immune regulation with regeneration (). This duality elevates the promise of Treg therapies—not only to suppress pathogenic immune responses but also to repair tissue damage left in their wake. These functions are governed by distinct molecular programs, enabling interventions that enhance regeneration without compromising immune control. While TCR stimulation drives classical suppressive activity, tissue-repair pathways can be activated independently by pro-inflammatory cytokines such as the alarmins IL-18 and IL-33, offering discrete levers for product precision (104). Harnessing this dual functionality through selective therapeutic tuning could improve outcomes in autoimmune disease by coupling tolerance restoration with repair, positioning Tregs as versatile living therapeutics for regenerative medicine.
AREG, an epidermal growth factor receptor (EGFR) ligand, acts as a master regenerative signal and is central to Treg-mediated tissue repair (105). In models of acute skeletal muscle injury, Tregs activate satellite cells to promote repair in an AREG-dependent manner; when Tregs are depleted, recovery falters and inflammation persists (106). This reparative function operates independently of immune suppression: selective deletion of AREG in Tregs has no effect on Treg suppressive activity but fails to prevent severe tissue damage (104). Beyond muscle, AREG-producing Tregs promote lung recovery after influenza (104), enhance cutaneous wound healing (107), and facilitate CNS regeneration (108), positioning Tregs as broad mediators of repair.
In addition to AREG, Tregs support repair through metabolic regulators such as PPARγ and secreted factors, including keratinocyte growth factor (KGF) in the lung (109) and nidogen-1 and secreted protein acidic and rich in cysteine (SPARC) in the heart (110). In the CNS, Treg-derived nerve growth factor and osteopontin reprogram microglia and promote oligodendrocyte differentiation, driving neuronal remyelination (111, 112). In the skin, Tregs accelerate wound repair through the AREG–EGFR axis and stimulate hair follicle regeneration via Notch signaling, which promotes hair follicle stem cell proliferation and differentiation (113). Collectively, these findings show that Tregs deploy diverse growth and metabolic factors to orchestrate repair, creating environments that promote regeneration and restore homeostasis. These programs are directed by the same transcriptional networks that optimize Tregs for tissue-specific tolerance, underscoring the need to achieve delivery precision by ensuring the therapy can adapt to its tissue context. For example, GATA3 enables skin Tregs to sustain AREG expression and drive hair follicle regeneration (114).
Tregs also modulate the sensory dimension of injury,—i.e., pain itself. While their anti-inflammatory activity provides indirect analgesia, a more direct mechanism has emerged in preclinical studies: Tregs secrete proenkephalin (Penk), the precursor of endogenous opioid peptides that act on local neuronal receptors to blunt nociception. Like their regenerative programs, this analgesic function operates independently of classical immune suppression and depends on glucocorticoid receptor signaling. Eliminating Penk expression specifically in Tregs leaves their suppressive ability intact yet markedly increases pain sensitivity in otherwise uninjured mice (115).
Penk-expressing Tregs are strategically positioned at neuroimmune interfaces, where they calibrate pain thresholds in a context-specific manner. In the skin, depletion of resident Tregs exaggerates thermal sensitivity, suggesting that tonic enkephalin release sets baseline heat-pain tolerance (115). In the meninges, a distinct Treg population modulates mechanical nociception, selectively dampening pressure and stretch signals, with effects most pronounced in females (116). This modality- and sex-specific regulation extends the spatiotemporal code of tolerance into sensory control. Although still untested in humans, the implication is striking: Tregs may act as sentinels of pain homeostasis, offering a blueprint for cell-based analgesic strategies that could reduce dependence on conventional opioids.
The role of Tregs as guardians of tissue homeostasis and repair thus extends far beyond immune suppression. Their regenerative polypharmacy offers multiple levers that can be used to sustain or restore tissue integrity. In autoimmune disease, next-generation Treg therapies could not only quell inflammation but also rebuild damaged tissues; in degenerative conditions not typically viewed as immune-mediated, their reparative capacity could slow decline or even restore lost function. This dual-function biology calls for an evolution in therapeutic vision—from tolerance medicine to regenerative tolerance medicine—where success is defined not merely by disease control but by the re-establishment of the spatiotemporal networks that sustain health.
Treg stability
Despite their potency, the suppressive and regenerative functions of Tregs remain vulnerable to stresses that erode tolerance and repair (117, 118). Persistent inflammation is the clearest example: proinflammatory cytokines such as IL-6 (119), IL-12 (120), and IL-1β (121) can destabilize the Treg transcriptional program, driving their conversion into “ex-Tregs”. These cells may retain FOXP3 expression but acquire effector-like traits, including secretion of interferon-γ (IFN-γ) and IL-17, amplifying rather than resolving inflammation. In some contexts, however, limited cytokine production by Tregs can aid tissue maintenance, such as by supporting epithelial integrity (122). This inversion of function underscores inflammation as a unifying pathology: the same spatiotemporal cues that normally imprint tolerance can, under chronic stress, corrupt it. Mechanistic dissection of this process has revealed therapeutic levers that could preserve both suppressive and regenerative identity, including reinforcement of FOXP3 stability (123) and protection from signals that drive conversion to ex-Tregs (124).
Aging constitutes a further axis of vulnerability. With advancing age, the suppressive and regenerative capacities of Tregs decline through impaired tissue communication and metabolic drift (125). In visceral adipose tissue, aged Tregs accumulate but fail to restrain insulin resistance (126), while in the CNS they lose adhesion molecules required for contact-dependent neuroregeneration, contributing to cognitive decline (127). Metabolically, aging Tregs shift from oxidative phosphorylation to glycolysis, diminishing both reparative and suppressive potential (128). These changes expose opportunities for intervention, such as engineering stabilizing circuits to resist inflammatory conversion or embedding metabolic programs that preserve repair capacity.
Inflammation and aging thus define dual frontiers for tolerance medicine, where restoring both arms of Treg function becomes a unified therapeutic goal. Understanding how these forces erode tolerance provides a roadmap for designing Treg therapies resilient to the very pressures that threaten them.
Environmental partners in the spatiotemporal code
Although the foundation of tolerance is written in our genetic code, it is not immutable. Genetics may author the blueprint, but the microbiome is its most prolific editor, continuously refining this program throughout life (129). This influence is evident in germ-free animals, which show impaired Treg development at barrier sites—a defect reversed when commensal flora are restored (130, 131). The effect is strongest in early life: recolonization during the neonatal period efficiently restores Treg populations, whereas later interventions are far less effective. This biology reframes the “hygiene hypothesis”. Rather than a simple absence of childhood infections, the more compelling “old friends” model proposes that modern environments lack the microbes and metabolites that co-evolved to imprint tolerance (132). Human data echo this imprinting: microbiome disruption in infancy, through antibiotics or cesarean delivery, correlates with higher risks of allergy, asthma, and autoimmunity (133). Together, these observations expose a paradox at the heart of tolerance: a system evolved to defend the self depends on dialogue with the non-self.
At the molecular level, metabolites mediate this dialogue, acting as dials that fine-tune differentiation and stability. The gut microbiome imprints Tregs through such signals, providing a general mechanism by which commensals program tolerance (134). Metabolites act both directly and indirectly. SCFAs produced by anaerobic fermentation of dietary fiber, such as butyrate and propionate, increase FOXP3 expression and histone acetylation at its locus, enhancing transcription and stabilizing lineage identity—direct evidence that microbial metabolites can epigenetically reprogram immune cells (82). They also indirectly restrain inflammation by reprogramming myeloid cells (135). Similarly, microbial conversion of bile acids into secondary forms skews dendritic cells toward an anti-inflammatory phenotype, which promotes differentiation of gut-specific RORγt+ Tregs (84). Paralleling the dual action of SCFAs, microbial catabolism of tryptophan generates aryl hydrocarbon receptor (AhR) ligands that both directly enhance the function of AhR+ Tregs (136) and induce IL-22 and IL-10 production by APCs and innate lymphoid cells, creating an anti-inflammatory milieu that supports Treg stability and accumulation (137–140). Collectively, these pathways establish the microbiome as a natural source of tolerance-promoting metabolites and a tangible lever for next-generation Treg therapies (141).
While the gut illustrates the importance of spatial imprinting, tolerance is equally shaped by temporal factors. Weaning illustrates this most vividly: the transition from milk to solid food introduces a surge of new antigens and microbial diversity (142). The rise of fiber-fermenting microbes boosts SCFA production, which stabilizes FOXP3 expression in gut Tregs and establishes durable intestinal tolerance (143). Similar early-life imprinting occurs elsewhere. During delivery, exposure to maternal vaginal microbes seeds the neonatal skin with long-lived Tregs (144, 145)—a process whose absence in cesarean births may contribute to elevated risks of allergy and autoimmunity (146). Lung development during infancy offers another temporal window: stress on the neonatal alveolar epithelium from first breaths, infection, or environmental exposure triggers alarmins IL-18 and IL-33, initiating reparative programming (147, 148).
Puberty marks another inflection point, as rising sex hormones broadly reprogram the immune system, reshaping inflammatory and regulatory pathways and contributing to the emergence of autoimmune susceptibility during adolescence (149). This transition coincides with the onset of the well-documented sex bias in autoimmunity, with women exhibiting higher incidence across most autoimmune diseases (150). Incidence curves for MS (151) and rheumatoid arthritis (RA) (152) diverge after puberty, suggesting that immune imprinting during this period influences disease risk later in life. Pregnancy represents another decisive window, during which the maternal immune system must tolerate the semi-allogeneic fetus. Treg populations expand dramatically, peaking during the second trimester, and inadequate expansion is linked to complications such as miscarriage and pre-eclampsia (153). While not as foundational as neonatal imprinting, both puberty and pregnancy constitute critical tolerance windows; reinforcing Treg function during these periods could mitigate autoimmune risk and prevent pregnancy-related immune complications.
The challenge now lies in translating these biological insights into robust interventions. Microbiome-based strategies—fecal transplantation, probiotics, prebiotics, and dietary changes—have shown promise but remain inconsistent (154, 155). More precise approaches, such as administering defined microbial metabolites or engineered commensals, offer greater control but are still emerging (156). Moving forward, tolerance medicine will require deliberate modulation of the hormonal, metabolic, and developmental transitions that shape immune balance across the lifespan. Timely interventions, such as reinforcing Tregs at puberty, sustaining tolerance during pregnancy, or preserving it with age, could transform transient vulnerabilities into therapeutic opportunities. The next decade must focus on converting environmental and temporal cues into durable, programmable tolerance. For tolerance medicine to become a reality, these insights must yield interventions as predictable and standardized as any drug: defined microbial consortia, engineered probiotics, metabolite therapies, and other tools to stabilize immune balance during decisive developmental windows.
Treg-based treatment strategies and technologies
From discovery to translation: building the era of tolerance medicines
Seven decades of research have brought the field to the threshold of a new era in which a deep understanding of Treg biology is transforming tolerance from theory to therapy (Figure 1). The journey began in the 1950s when Medawar, Burnet, and Owen first demonstrated acquired tolerance (157). In the decades that followed, studies showed that tolerance could be transferred with leukocytes from tolerant animals, pointing to a specialized cell population capable of enforcing immune restraint.
Early enthusiasm was tempered by decades of skepticism. Inconsistent results and the absence of defining markers obscured the identity of these “suppressor T cells”. This impasse was finally broken by the discoveries recognized by the 2025 Nobel Prize in Physiology or Medicine, which honored the identification of FOXP3+ Tregs as the cellular foundation of immune tolerance (). Sakaguchi’s landmark finding that depletion of CD4+CD25+ T cells triggers fatal autoimmunity provided the first functional proof (158), while the subsequent identification of FOXP3 as the lineage-defining transcription factor by Drs. Fred Ramsdell, Shimon Sakaguchi, and Alexander Rudensky in the early 2000s supplied the molecular key that ended decades of controversy (159–161). Mutations in FOXP3 cause catastrophic autoimmunity in mice (“scurfy”) (162) and humans (immunodysregulation polyendocrinopathy enteropathy X-linked, or IPEX, syndrome; 163, 164), revealing that a remarkably small population of T cells serve as the indispensable architects of immune homeostasis and self-tolerance.
With these foundations established, Tregs are now recognized as the keystone of peripheral tolerance, opening a therapeutic frontier in which they can be isolated, expanded, and deployed as living drugs to restore immune balance and tissue health. Preclinical studies in the early 2000s laid the groundwork for human translation: adoptive Treg transfer in mice mitigated inflammation and tissue injury across diverse settings, including graft-versus-host disease (GvHD) (165), inflammatory bowel disease (166), T1D (167), experimental autoimmune encephalomyelitis (168), collagen-induced arthritis (169), organ transplant rejection (170), and even non-immune conditions such as stroke (171) and amyotrophic lateral sclerosis (ALS) (172). Collectively, these studies marked the dawn of the precision tolerance restoration paradigm: a shift from blunt immunosuppression to the strategic reprogramming of the immune system to reinstate long-term equilibrium (Figure 5; Supplementary Table 1).
Figure 5
Clinical experience of first-generation polyclonal Tregs
First-generation Treg therapy trials utilized ex vivo–expanded, unmodified, polyclonal Tregs to treat transplantation complications and autoimmune disease (173, 174). Success in these pioneering studies depended on key advances in Treg isolation and manufacturing (175). Early trials established an excellent safety profile, showing no evidence of global immunosuppression or toxicities typical of Teff therapies—such as cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS)—and bypassed the need for lymphodepleting preconditioning ().
Clinically, these therapies have demonstrated the potential to reduce immunosuppression requirements in kidney (176) and liver (177) transplant recipients, improve bone marrow engraftment and reduce acute GvHD (178), and preserve β-cell function in recent-onset T1D (174, 179). However, these studies also underscored the challenges of converting a “regulatory cell” into a good manufacturing practice (GMP)-grade medicinal product. Unlike other lymphocytes, Tregs are inherently fragile, demanding subset-specific measures to maintain product quality. Ensuring a stable regulatory phenotype and managing sensitivity to cryopreservation remain critical hurdles in maintaining product quality (180, 181).
In these early trials, efficacy was heavily dependent on disease stage. Most studies were phase I (some expanded to I/IIa) and enrolled patients with advanced disease (). Achieving restitutio ad integrum—a full return to health—is unlikely in chronic indications such as GvHD with established fibrosis, advanced MS with neurodegeneration, or stage 3 T1D following recurrent hyperglycemia. Consequently, more sustained clinical effects have emerged when polyclonal Tregs were applied prophylactically. This is evidenced by the success in preventing GvHD (182) or treating T1D patients while viable β-cells remained (179). In the ONE study of polyclonal Tregs in newly transplanted living-donor kidney recipients, several trial arms reported reduced requirements for immunosuppression (176), and high-resolution spatial profiling of biopsies revealed that lymphocytic infiltrates harbor a distinct immunoregulatory signature, suggesting that local tissue regulation is a key driver of long-term stability (183).
However, the follow-up TWO study suggests that early intervention may not always be necessary; a delayed 6-month infusion protocol achieved 100% rejection-free survival and facilitated easier logistics through long-term cryopreservation (184). These data reinforce the importance of the spatiotemporal aspects of tolerance and demonstrate that therapeutic windows are dynamic targets, where successful immune re-equilibration depends on aligning the timing of delivery with the specific immunologic context and stage of disease.
Beyond adult peripheral blood, alternative biological sources of polyclonal Tregs are being harnessed to overcome manufacturing bottlenecks and improve product quality. Autologous tTregs, routinely harvested during pediatric cardiac surgery, have demonstrated high purity and the capacity to safely restore the Treg pool for at least 2 years post-heart transplant (185). The high cell yield and stable phenotype of these “naive” cells facilitate the establishment of a durable regulatory population, even in the complex inflammatory environment of a cardiac allograft.
While engineering offers new capabilities, the simpler manufacturing of polyclonal products remains a significant advantage. In contexts such as organ transplantation, where the frequencies of alloreactive Tregs are high and the bystander effect can be leveraged, these foundational products may suffice to establish durable tolerance. Collectively, these efforts have advanced the field to regulatory recognition, as the United States Food and Drug Administration recently started reviewing the first Biologics License Application for a polyclonal Treg product with the Prescription Drug User Fee Act (PDUFA) date soon (186*), marking the transition from experimental therapy to clinical reality (187).
However, the limitations of first-generation products, particularly regarding potency in advanced disease and tissue-specific persistence, have set the stage for next-generation approaches. Advances in targeted engineering now allow disease-specific modulation, extending therapeutic reach to later disease stages and enabling treatment of even advanced conditions with purpose-designed, engineered Tregs (Figure 6).
Figure 6
Clinical experience using engineered Tregs
Antigen-specific Tregs
A growing body of evidence demonstrates the enhanced efficacy of receptor-engineered Tregs, reinforced by recent clinical data (188). While polyclonal Tregs are foundational, CAR- and TCR-engineered Tregs offer the distinct advantage of directing suppression to disease-relevant tissues (189–191). In preclinical models, TCR-engineered Tregs have been successfully redirected toward self-antigens (), commensal antigens on gut APCs (192), and stress-induced self-peptides (193). Complementing this is the use of CARs, which fuse antigen-specific single-chain variable fragments (scFvs) with potent intracellular signaling domains (194). By combining precise specificity with integrated costimulation, CARs allow Tregs to bypass traditional major histocompatibility complex (MHC) restriction to focus regulatory activity exactly where it is needed.
Beyond these established platforms, the engineering toolkit is expanding to improve both safety and functional reach. Non-viral clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas)12a editing now enables the integration of antigen-binding domains directly into the endogenous TCR/CD3 complex. These “TRuC-Tregs” benefit from physiological signaling and preferential enrichment while mitigating the insertional risks associated with retroviral gene transfer (195). Furthermore, the scope of precision is extending to the humoral arm of immune rejection. Chimeric anti-HLA antibody receptors (CHARs) utilize extracellular HLA proteins to directly engage and suppress alloreactive B cells, providing a novel path for desensitizing transplant recipients who harbor pre-existing anti-HLA antibodies (196). Together, these advances transition Tregs from broad immune modulators to precision therapeutics capable of selectively neutralizing pathogenic responses or restoring immune homeostasis within specific clinical contexts.
Several antigen-specific engineered Treg therapies have now entered clinical testing. First-in-human studies using HLA-A2 CAR-Tregs are evaluating antigen-specific regulation in kidney and liver transplantation (197, 198) and CD6 CAR-Tregs after allogeneic hematopoietic cell transplantation (199). In autoimmune disease, CAR-Treg trials are underway in RA [United States national clinical trial (NCT) NCT06201416] and hidradenitis suppurativa (NCT06361836), with encouraging early results (188). Tregs engineered to express autoantigen-specific TCRs are also being tested in MS, celiac disease, and T1D. In parallel, investigators are exploring an alternative strategy: reprogramming conventional CD4+ T cells with ectopic FOXP3 expression and disease-specific TCRs, effectively converting them into Treg-like cells for T1D therapy (NCT06919354).
Using synthetic biology to enhance Treg functionality
Beyond refining antigen specificity, synthetic biology offers a toolkit for the multifaceted enhancement of Treg functionality. Efforts focus on engineering tissue-homing receptors to more precisely direct cells to disease sites (103, 200). Once there, inducible circuits responsive to environmental cues or small molecules are being developed to modulate metabolism, trigger cytokine or repair factor release, or act as rheostats that fine-tune activity—improving both safety and adaptability (201, 202).
Logic-gated CARs introduce an additional layer of control: AND gates activate Tregs only when both an autoantigen and a tissue-specific marker are present, preventing off-target suppression, while NOT gates terminate activation in response to tumor antigens, limiting immune evasion (203). Collectively, these innovations aim to transform Tregs into “Trojan horses” that infiltrate diseased tissues and deliver therapeutic payloads with spatial and temporal precision, moving from targeted suppression to true immune engineering.
Using synthetic biology to enhance Treg stability and persistence
Delivery alone is insufficient; Tregs must persist and maintain their lineage identity within hostile, inflammatory microenvironments. Teff, macrophages, and cytokines such as IL-6 and tumor necrosis factor α (TNF-α) can destabilize Tregs by suppressing FOXP3 expression, risking conversion into effector-like cells that exacerbate rather than resolve inflammation. Because sustained function is essential for tolerance, instability remains a key obstacle to effective Treg therapy. Overcoming this vulnerability has become a central goal of next-generation engineering strategies.
Multiple strategies are being explored to reinforce Treg fidelity in disease. FOXP3 stability can be enhanced through overexpression (204, 205), epigenetic modulation of the Treg-specific demethylated region (TSDR) (206), or co-expression of stabilizing factors such as Helios (207). Ectopic FOXP3 expression in both naive T cells and Tregs is also under investigation (208). Because Tregs rely on IL-2 for survival and proliferation, engineered receptors and circuits are being developed to provide autonomous signaling in its absence (209). Synthetic circuits that trigger the release of immunoregulatory cytokines during inflammation aim to sustain activity even when FOXP3 is transiently destabilized (210). Novel synthetic promoters are likewise being developed to ensure that key transgenes, such as CARs, are expressed only in stable, bona fide Tregs (211). Together, these innovations aim to extend in vivo persistence and ensure the “orchestrator of tolerance” does not become a driver of damage.
Beyond intrinsic engineering, Treg stability also depends on cues from the tissue environment, from access to critical growth factors such as IL-2 to cellular and metabolic support—an area now primed for therapeutic exploration. Crosstalk with other cells provides essential reinforcement: tolerogenic dendritic cells (tolDCs) present antigen and secrete cytokines such as IL-10 and TGF-β, sustaining FOXP3 expression and suppressive function (212). In addition to producing TGF-β and expressing IDO, stromal cells can “donate” mitochondria to Tregs, enabling them to withstand metabolic stress (213). Metabolic cues likewise shape Treg stability, from fatty acid-rich niches that support oxidative metabolism to microbiota-derived metabolites that lock in lineage fidelity. Ultimately, durable success will require integrating intrinsic engineering with environmental signals that render tissues more Treg-permissive, establishing the foundation for next-generation, Treg-friendly therapies and synergistic combinations.
Combination approaches
As with cancer and other immune-mediated diseases, the efficacy of Treg therapy in patients with high disease burden may depend on combination strategies that enhance Treg activity or remove barriers to effectiveness. Early-phase trials have shown that both low-dose wild-type IL-2 and IL-2 muteins can selectively expand Tregs (214). In one study, low-dose IL-2 was combined with polyclonal Tregs for recent-onset T1D: while IL-2 administration increased infused Treg numbers, it also expanded a small subset of CD8+ Teffs, limiting its beneficial effect (215). In a liver transplantation study, low-dose IL-2 expanded circulating Tregs but did not promote allograft tolerance (216). Next-generation agents such as IL-2 muteins (217) and TNFR2 agonists (218) and even some immunosuppressive therapies such as rapamycin—which selectively expands Tregs, promotes tolDCs, and restrains effector activity—offers additional avenues for synergy. Biomaterials and scaffolds present another opportunity, enabling co-delivery of Tregs with IL-2 or other supportive factors directly to diseased tissues to promote local engraftment and expansion (219). Finally, combination approaches can reinforce tolerance networks by recruiting complementary regulatory populations including tolDCs, type 1 regulatory (Tr1) T cells, mesenchymal stem cells, and other immune partners that stabilize systemic immune balance (220).
Conversely, strategies that transiently inhibit or deplete effector cells can optimize the Treg-to-effector ratio and enhance therapeutic impact. Examples include Teff-targeting agents such as anti-thymocyte globulin (ATG) (221), the anti-CD3 antibody, teplizumab (222), and newly developed anti-CD6 (223) and anti-CD2 (224) antagonists, which selectively eliminate activated Teffs and natural killer (NK) cells. Tregs have been engineered to resist calcineurin inhibitors that otherwise impair their function, allowing Treg therapy to be paired with standard immunosuppression in transplantation (225, 226). In another study, efficient B cell depletion enhanced the regulatory effects of adoptively transferred Tregs in T1D (likely by removing APCs) and outperformed Treg monotherapy (227). Other agents may act synergistically by alleviating immune dysregulation, enabling inflammation-recruited Tregs to more effectively restore immune equilibrium (228). Collectively, these findings underscore a clear priority for the field: durable tolerance will require rational combination strategies that extend Treg persistence, reinforce function in inflamed tissues, and coordinate regulatory and effector control.
Expanding access: in vivo and allogeneic approaches
The potential of Treg therapies remains constrained by a major hurdle: the need to manufacture individualized products for each patient. Autologous cell therapy remains the most reliable means of ensuring immune acceptance of infused cells, but the process is complex, time-consuming, and costly, limiting broad clinical adoption. The promise of tolerance medicine will only be realized when access extends beyond bespoke manufacturing through the development of universal donor platforms and in vivo reprogramming strategies capable of delivering tolerance at scale.
One path forward involves the development of “off-the-shelf” allogeneic therapies, produced in large batches and ready for immediate administration. To succeed, these cells must be truly universal—capable of evading recognition from diverse immune systems. Two main strategies are being pursued: deriving allogeneic Tregs from healthy donors or differentiating them from induced pluripotent stem cells (iPSCs). Healthy-donor Tregs offer the advantage of starting with mature, functionally validated cells; studies have shown CRISPR-edited hypoimmunogenic (HLA class I and II knockout; HLA-E knock-in) Tregs evade attack by T and NK cells while retaining FOXP3 stability and suppressive activity (229). However, the cumulative loss of editing efficiency with sequential gene edits remains a challenge with this approach (230).
In contrast, the iPSC route shifts the engineering burden to the pluripotent stage, allowing for complex, multi-locus modifications before differentiation into functional iPSC-derived Tregs (231, 232). Although in vitro differentiation remains complex and may not fully recapitulate thymic cues that guide endogenous Tregs (233), this strategy offers a powerful advantage: a single correctly edited stem cell could, in principle, yield an unlimited supply of standardized, precisely engineered Tregs. In both systems, further safeguards are needed to prevent immune rejection, including gene editing to silence allogeneic MHC expression (234) and molecular “shielding” through CD47 (235, 236) or ligands that engage NK-cell inhibitory receptors (237).
The ultimate solution may lie in engineering Tregs directly in vivo. Instead of complex ex vivo manufacturing, Tregs could be programmed within the patient using viral or non-viral delivery systems designed for in situ reprogramming. These targeted platforms are in early development but show remarkable promise. Rather than broad reprogramming, transgenes can be placed under the control of small synthetic promoters derived from the FOXP3 or CD25 loci to restrict expression specifically to Tregs (211). Lipid nanoparticles functionalized with antibodies or ligands can deliver DNA or RNA encoding CARs and other payloads, directing Tregs to specific tissues while increasing precision and minimizing off-target effects (238, 239). In some settings, direct in vivo programming may prove more practical than allogeneic cell therapy, particularly as recent gene therapy approvals establish regulatory precedents. Regardless of the approach, scalable and accessible treatments are urgently needed to reach underserved patients and to enable rapid intervention in acute, time-critical settings.
As the field transitions from foundational discovery to clinical implementation, the developmental landscape reflects a diverse array of platforms at varying stages of maturity (Table 1). Understanding these technical and regulatory benchmarks provides the necessary context for evaluating the application of Treg therapies across specific clinical indications, where successful outcomes depend on matching the appropriate platform to the unique immunologic requirements of each disease.
Table 1
| Modality | Indication | Key strengths | Major risks/ open issues | Current clinical stage |
|---|---|---|---|---|
| Polyclonal ex vivo expanded Tregs | • T1D • GvHD • Transplantation | • Established safety profile • Manufacturing experience • Leverages bystander suppression | • Dose requirements • Persistence • Potency • Variability | Phase I and II |
| Antigen-specific (TCR or TCR-enriched) Tregs | • T1D • MS • Celiac disease • Transplantation | • Increased potency per cell • Targeted suppression | • Antigen identification • HLA restriction • Epitope spreading | Early phase I |
| CAR-engineered Tregs | • T1D • IBD • GvHD • RA • Transplantation • MS • ALS | • MHC-independent targeting • Modular design • Tissue-targeting potential | • Stability under activation • Off-target effects • Persistence control | Early phase I |
| Allogeneic edited “off-the-shelf” Tregs | • T1D • IBD • GvHD • Transplantation • MS • ALS | • Scalability • Rapid availability | • Host rejection • Gene editing safety • Regulatory complexity | Pre-clinical/Early phase I |
| In vivo gene-modified or reprogrammed Tregs | • MS • Transplantation • IPEX • Broad autoimmune applications | • Avoids ex vivo manufacturing • Potential scalability | • Controllability • Insertional mutagenesis • Durability; oncogenic risk | Discovery |
Clinical readiness of regulatory T cell (Treg) therapy modalities.
Abbreviations: ALS, amyotrophic lateral sclerosis; CAR, chimeric antigen receptor; GvHD, graph-versus-host disease; HLA, human leukocyte antigen; IBD, inflammatory bowel disease; IPEX, immunodysregulation polyendocrinopathy enteropathy X-linked syndrome; MHC, major histocompatibility complex; MS, multiple sclerosis; RA, rheumatoid arthritis; T1D, type 1 diabetes; TCR, T cell receptor.
Emerging frontiers
Infection-induced immune dysfunction and acute tissue injury conditions
Chronic infections and acute viral illnesses exemplify the “double-edged sword” of tolerance: excessive regulation permits pathogen persistence, whereas insufficient control drives immunopathology. In chronic infections such as HIV (240), hepatitis B virus (241), and hepatitis C virus (242), Tregs accumulate in tissues and suppress cytotoxic CD8+ T-cell responses, contributing to viral persistence. Depletion studies in animal models can restore viral control but often at the cost of severe tissue damage (243). Advances in single-cell and spatial profiling are now mapping the cellular circuits that sustain pathological tolerance in chronic infection, revealing opportunities for selective intervention (244). Targeted disruption of suppressive networks or in situ reprogramming of Tregs could restore pathogen control without provoking harmful inflammation.
The COVID-19 pandemic revealed the opposite extreme, where mortality often resulted not from viral replication but from a collapse of tolerance, leading to uncontrolled inflammation (245). Similar breakdowns occur in other acute settings, including sepsis (246), CRS (247), and immune effector cell-associated neurotoxicity syndrome (248) observed with effector CAR-T cell therapies. Therapeutic intervention in these settings is limited by the time required to manufacture autologous Tregs, underscoring the need for allogeneic products that can be deployed rapidly. Although their persistence may be limited, short-lived allogeneic Tregs could still provide meaningful benefit. Early studies investigated umbilical cord-derived allogeneic Tregs in patients with COVID-19-associated acute respiratory distress syndrome (ARDS), leveraging their naive phenotype and reduced alloreactivity for rapid, off-the-shelf deployment (249). While still preliminary, these trials mark an important milestone for tolerance medicine, demonstrating that Treg therapy might be mobilized quickly to blunt hyperinflammation and limit acute tissue injury in real time. Together, these examples point to a broader goal. The next challenge is to develop tolerance-based interventions that can be deployed rapidly and safely in acute settings, extending the reach of Treg therapy from chronic disease to immediate, life-threatening conditions.
Breaking tolerance in cancer
In some contexts, breaking tolerance is as important as enforcing it. The same regulatory circuits that prevent autoimmunity can be hijacked in cancer, where Tregs suppress antitumor immunity and allow malignant cells to persist (250). Addressing this “dark side” of tolerance is inherently challenging: disrupting regulation can restore immune attack but risk self-reactivity. With their command of physiological tolerance mechanisms, Treg-based strategies may ultimately enable precise control of this balance.
Across solid tumors, high intratumoral Treg infiltration correlates with impaired Teff responses and poor prognosis, identifying Tregs as active enablers of immune escape (251). The tumor microenvironment provides a Treg-permissive niche enriched in chemokines that recruit Tregs (252) and soluble factors such as lactate that sustain them—enhancing expansion, survival, and fitness over effector cells (253). Within this milieu, tumor-infiltrating Tregs express high levels of co-inhibitory molecules that directly suppress effector activity, secrete TGF-β and IL-10 to dampen immunity and promote tolerogenic myeloid populations, and consume IL-2 while generating adenosine to exert metabolic control (254). Thus, regulatory pathways that normally preserve tissue integrity are co-opted to promote tumor persistence.
These insights have inspired a range of strategies to deplete or disable Tregs, including immune checkpoint inhibitors, such as the CTLA-4-targeting antibody ipilimumab (255) and programmed cell death protein 1 (PD-1)-blocking antibody pembrolizumab (256), as well as emerging chemokine receptor antagonists targeting CCR4 (257) and CCR8 (258–260) and agents that remodel the microenvironment (261). Although treating “over-tolerance” with additional Tregs may seem paradoxical, advances in cell engineering now make it possible to reprogram them to act selectively within tumors. Because tumor cells actively recruit Tregs, these cells can be transformed into “Trojan horses” engineered to deliver payloads that disrupt local suppression or carry synthetic circuits that convert inhibitory cues into localized immune activation, repurposing tolerance itself as a precision weapon against cancer.
“Non-immune” indications
The same mechanisms that make Tregs powerful tools for restoring immune balance may also unlock therapies for conditions not traditionally viewed as immunological. Inflammation is now recognized as a common driver or amplifier of nearly every major disease category (262). This realization positions tolerance as a unifying principle that could guide more integrated therapeutic strategies across medicine.
Neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS) remain among the most intractable challenges in medicine. Yet even within the nominally immune-privileged CNS, Tregs infiltrate sites of injury and engage with glial and neural precursor cells to restrain inflammation and promote repair (263, 264). Rather than acting directly on disease triggers, they recalibrate the local environment—slowing, and in some cases partially reversing, neuronal loss and cognitive decline. Building on these preclinical findings, early clinical trials are now testing whether Treg infusion or Treg-supportive interventions such as low-dose IL-2 can attenuate neuroinflammation and modify disease progression (265).
Metabolic and cardiovascular diseases further demonstrate how dysregulated tolerance drives chronic inflammation. Atherosclerosis and metabolic syndrome are increasingly recognized as fundamentally inflammatory disorders rooted in aberrant environmental and metabolic cues, highlighting the role of vascular and adipose Tregs in maintaining homeostasis (266, 267). In atherosclerosis, regulatory mechanisms become progressively overwhelmed by proinflammatory cells within plaques, sustaining vascular inflammation and driving plaque growth (268). In adipose tissue, chronic low-grade inflammation promotes insulin resistance and progression to diabetes (269), while loss or dysfunction of adipose Tregs is linked to obesity and metabolic syndrome (270, 271). By restoring immune equilibrium, Treg-based therapies could offer a new strategy for managing these widespread diseases.
The reparative functions of Tregs open new frontiers for regenerative medicine. Persistent inflammation can distort normal repair processes, leading to excessive scarring and fibrosis that compromise organ function (272). By tempering immune activation and releasing factors that promote tissue remodeling, Tregs support orderly repair and recovery—properties that could be harnessed to enhance healing. Yet this same biology can cut both ways: when subjected to chronic injury or dysregulated signaling, reparative Tregs may amplify fibroblast activation and drive pathological fibrosis (273). In such contexts, precision approaches will be essential, requiring selective engagement of pathways that restrain inflammation and guide constructive remodeling while directing Tregs toward resolution rather than scarring. Achieving this level of control would extend the promise of tolerance from maintaining immune health to preserving tissue integrity across the lifespan.
Tregs may also be harnessed to counter the effects of aging, which are increasingly recognized as a state of chronic, low-grade inflammation. This persistent immune activation contributes to neurodegeneration, atherosclerosis, frailty, and impaired wound healing (274). Current interventions such as anti-inflammatories, antioxidants, and senolytics target individual pathways but leave the underlying imbalance unresolved (275). In contrast, Tregs recalibrate tolerance more broadly, dampening chronic inflammation while sustaining repair. Harnessing these properties will be key to translating tolerance biology into interventions that sustain repair and preserve resilience, thus extending both life and healthspan.
Limitations and open questions
Despite the broad potential of harnessing the innate abilities of Tregs, there are scientific, technical, and clinical uncertainties that must be addressed to transition from proof-of-concept to a durable therapeutic platform and to keep patient safety at the forefront of next-generation therapies. Addressing these uncertainties will require multidisciplinary innovation in all areas, including biological characterization, cell engineering, manufacturing, and clinical trial design. Standardization and harmonization across Treg therapies will also be crucial over the coming years to ensure a robust foundation for expansion into new indications.
To ensure long-term safety, Treg stability and lineage fidelity in inflammatory environments are critical concerns. Although human and mouse data support the epigenetic stability of lineage-committed Tregs, robust cell division and inflammatory cytokines (e.g., IL-6 and IL-1β) can modulate their phenotype and function (103, 276, 277). Accordingly, in contrast to CAR-T cells, moderate antigen receptor stimulation may be ideal for preserving Treg stability (278). The long-term stability of ex vivo-expanded, engineered Tregs in highly inflamed tissues remains incompletely defined and is still being investigated in ongoing clinical studies. Early clinical evidence suggests that CAR-Tregs can migrate to inflamed tissues, maintain suppressive potential without causing toxicity, and demonstrate signs of tissue engraftment and persistence (199). Longitudinal studies and standardized functional assays will be needed to verify that infused Tregs maintain their suppressive identity over time.
Although engineering specificity aims to localize suppression, many conditions involve poorly defined antigenic drivers or shifting epitope profiles. Consequently, a “one-size-fits-all” approach may not be the optimal path to Treg therapeutic success. In some cases, a broad polyclonal Treg approach may be more appropriate for indications not associated with specific autoantigens (e.g., in transplantation), whereas, in well-defined disease settings, antigen-specific approaches ensure that the right autoantigens are targeted. As demonstrated in human T1D studies, antigen-specific Tregs can be successfully expanded in vitro; however their suppressive hierarchy and epigenetic stability are dictated by the specific nature of the antigenic challenge (279). This complexity reinforces the need to move beyond monolithic strategies, selecting the right approach based on the spatiotemporal and kinetic characteristics of disease.
Transitioning from lab-scale to industrial-scale Treg therapeutics will require significant technical innovation. Ex vivo expansion and engineering introduce significant manufacturing complexities, although it is hoped that next-generation in vivo approaches may eventually circumvent these. The immediate strategic priorities, however, remain ensuring reproducibility, defining standardized potency metrics, and streamlining manufacturing. Collaborative initiatives, such as the Minimum Information about T Regulatory Cells (MITREG) checklist (280), are essential to drive the cell processing, quality control, and regulatory harmonization required for global scalability.
While clinical trials have not yet yielded significant safety signals, the transition to next-generation approaches introduces new liabilities. As the field moves toward genetic modifications that increase the durability of these cells, the inherent oncogenic and insertional risk also increases. Strategies such as targeted gene insertion (281, 282), safety switches (283), regulatable promoters (284), and transient delivery platforms (285) will mitigate these technical concerns and provide a necessary layer of protection for engineered products.
Ensuring safety also extends beyond technical controllability. Due to the spatiotemporal nature of tolerance, the context of tolerance restoration must be considered. Durable tolerance is desirable in autoimmunity and transplantation but potentially hazardous in the context of occult malignancy or chronic infection; effective suppression of a localized autoimmune driver could inadvertently create a permissive environment for tumor growth or viral escape. Crucially, this theoretical risk must be weighed against the proven infectious and oncogenic burdens of current systemic agents, which the ONE study suggests may be mitigated through Treg-mediated drug weaning (176). Overall, the risk–benefit ratio success of Treg therapies depends not only on the cell product itself but also on a sophisticated understanding of the local tissue milieu and the timing of intervention relative to disease stage.
Establishing definitive clinical endpoints and biomarkers is essential for refining Treg therapies and predicting individual patient responses. However, identifying robust markers of potency, tissue engagement, and mechanistic response is currently constrained by the ethical imperative to minimize invasive procedures. To shift from simple cell enumeration toward functional, tissue-level readouts, the field must prioritize innovation in non-invasive surrogates. Strategies capable of maximizing information from minimal samples, such as next-generation molecular profiling (e.g., cell-free DNA epigenetic signatures) (286) and advanced imaging techniques (e.g., immuno-positron emission tomography) (287) will be required to expand scientific insights while safeguarding patient well-being.
Taken together, these considerations underscore that Treg-based therapies represent a biologically compelling, yet technically demanding, modality. Addressing these open questions will determine whether tolerance restoration fulfills its promise as a transformative and durable therapeutic platform.
Roadmap to Treg-centric tolerance medicine
Autoimmunity and transplantation remain the primary proving grounds for tolerance-based therapies, where immune dysregulation is well defined. Early clinical trials have shown that Treg therapy is both safe and feasible. The lesson from these efforts is clear: deeper mechanistic insight, coupled with rapid technological innovation, now compels the development of next-generation strategies. Together, the biological and engineering advances outlined in this article provide the foundation to convert these early successes into durable, and potentially curative, tolerance.
To effectively advance tolerance medicine, multiple approaches must progress in parallel; achieving durable tolerance will likely require engaging with several dimensions of Treg biology. In the near term, rational combination strategies represent the most direct path to clinical impact, pairing Tregs with cytokine or immunomodulatory agents that enhance persistence and function or relieve effector pressure. These efforts can extend the durability of benefit in ongoing trials and provide the critical proof of principle needed to guide next-generation designs. As engineering, metabolic tuning, and tissue-targeting technologies mature, these elements can be layered onto combination platforms to reinforce lineage stability, leverage tissue-specialized programs, enhance homing and persistence, and amplify network-building capacity.
Tolerance medicine now stands at a critical juncture, as biology, technology, and clinical experience converge to transform a long-held aspiration into the foundation of a new medical paradigm. With sustained investment, cell-based therapies such as Tregs could become a new pillar of medicine alongside small molecules, biologics, and gene therapies, extending the reach of modern treatments to diseases once deemed incurable. The promise extends beyond patients: tolerance offers clinicians powerful new tools, provides industry with scalable platforms, and establishes clearer regulatory pathways, creating a framework with the potential to elevate the entire healthcare ecosystem.
To realize this vision, tolerance medicine will require a patient-centered compass that defines our current position and guides the path forward. Current measures of tolerance capture only fragments of a larger whole; integrated biomarkers will be essential to reveal what works, what does not, and why. From robust indicators of FOXP3 stability to multiomic and systems-level signatures encompassing the full breadth of Treg function, these tools will drive personalization, prediction, and regulatory confidence, ultimately advancing tolerance therapies from experimental interventions to a central pillar of modern medicine.
Tolerance medicine will not be universal in application or timing. Just as Tregs mature within defined developmental windows, diseases present their own critical moments for intervention, such as the early post-transplant period before anti-graft immunity takes hold. The success of these approaches will hinge on precision: the right Treg, delivered in the right way, to the right tissue, at the right time—laying the proof-of-principle foundation to extend tolerance medicine across human health and disease.
Strategic investment will be essential to move tolerance medicine from concept to reality through cross-disciplinary collaboration, regulatory partnership, and sustained funding. While early signs of clinical benefit are emerging, the next challenge is to scale development and ensure equitable access worldwide. With commitment and coordination, tolerance medicine can advance from aspiration to achievement, defining the next era of medical innovation.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fsci.2026.1792210/full#supplementary-material
Supplementary Table 1Interventional trials with Tregs. The data are from the following databases: ClinicalTrials.gov (https://www.clinicaltrials.gov), European Union Clinical Trials Register (https://www.clinicaltrialsregister.eu), CTIS (https://euclinicaltrials.eu/), ISRCTN—The UK’s Clinical Study Registry (https://www.isrctn.com), Chinese Clinical Trial Registry (https://www.chictr.org.cn), Clinical Trials Registry—India (https://ctri.nic.in), and KONECT Korea National Enterprise for Clinical trials (https://www.konect.or.kr) assessed between 18-20.10.202
Statements
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
Author contributions
JAB: Conceptualization, Writing – review & editing, Writing – original draft.
MKL: Conceptualization, Writing – review & editing, Writing – original draft, Visualization.
FJR: Writing – original draft, Writing – review & editing, Conceptualization.
AYR: Writing – original draft, Writing – review & editing, Conceptualization.
QT: Writing – original draft, Conceptualization, Visualization, Writing – review & editing.
PT: Writing – original draft, Conceptualization, Visualization, Writing – review & editing.
Funding
The authors declared that financial support was received for this work and/or its publication. PT received support for this study via PolTREG S.A. from the Medical Research Agency, Poland (grants no. 2022/ABM/05/00001-00 and no. 2024/ABM/05/KPO/KPOD.07.07-IW.07-0234/24). MKL is a Canada Research Chair in Engineered Immune Tolerance and receives a Scientist Salary Award from the BC Children’s Hospital Research Institute. The funders were not involved in the writing of this article or the decision to submit it for publication.
Conflict of interest
The authors declared that the preparation of the manuscript and some of the content could be construed as a potential conflict of interest with regards to Sonoma Biotherapeutics (South San Francisco, CA, United States) and PolTREG (Gdańsk, Poland).
AYR is a member of the Scientific Advisory Board of, and holds equity in, Sonoma Biotherapeutics, RAPT Therapeutics (South San Francisco, CA, United States), Coherus Oncology (Redwood City, CA, United States), Santa Ana Bio (Alameda, CA, United States), Odyssey Therapeutics (Boston, MA, United States), Nilo Therapeutics (New York, NY, United States), and Vedanta Biosciences (Cambridge, MA, United States). He is also a member of the Scientific Advisory Boards of Amgen (Thousand Oaks, CA, United States) and BioInvent International (Copenhagen, Denmark). He has been a consultant for Radionetics Oncology (San Diego, CA, United States) and AbbVie (North Chicago, IL, United States). AYR holds patents for Chemokine receptor 8 (CCR8)-based therapeutic depletion of tumoral Treg cells.
JAB is the previous CEO, and co-founder of, and holds equity in, Sonoma Biotherapeutics. JAB is also a member of the Scientific Advisory Board, and holds equity in, Arcus Biosciences (Hayward, CA, United States) and is a member of the Board of Directors and holds equity in Gilead Sciences (Foster City, CA, United States). JAB holds a number of patents from the University of California San Francisco (UCSF) and Sonoma Biotherapeutics on Treg cell therapy.
FJR is co-founder and a member of the Scientific Advisory Board of Sonoma Biotherapeutics, in which he holds equity.FJR is also a member of the Scientific Advisory Board, and holds equity in, Georgiamune, Inc. (Gaithersburg, MD, United States) and Equillium, Inc. (San Diego, CA, United States).
QT is co-founder and member of the Scientific Advisory Board of Sonoma Biotherapeutics, in which she holds equity. QT holds several patents from UCSF on Treg cell therapy.
PT is a co-inventor of patents in the Treg programs mentioned in this manuscript. PT is compensated by and holds equity in PolTREG.
MKL holds patents from the University of British Columbia on Chimeric Antigen Receptors for use in Tregs. She is a member of the Scientific Advisory Board of Quell Therapeutics London, United Kingdom.
The reviewer FI declared a past co-authorship with the authors MKL, QT, PT to the handling editor.
The authors QT, AR and PT declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.
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Summary
Keywords
autoimmunity, FOXP3, immune regulation, immune tolerance, Tregs, regulatory T cell
Citation
Bluestone JA, Levings MK, Ramsdell FJ, Rudensky AY, Tang Q and Trzonkowski P (2026) Regulatory T cells: master orchestrators of immune tolerance and tissue homeostasis. Front Sci 4:1792210. doi: 10.3389/fsci.2026.1792210
Received
20 January 2026
Revised
12 March 2026
Accepted
28 May 2026
Published
25 June 2026
Volume
4 - 2026
Edited by
Shohei Hori, The University of Tokyo, Japan
Reviewed by
Kathryn Wood, University of Oxford, United Kingdom
Fadi Issa, University of Oxford, United Kingdom in collaboration with reviewer KW
Ethan Menahem Shevach, National Institutes of Health (NIH), United States
Updates
Copyright
© 2026 Bluestone, Levings, Ramsdell, Rudensky, Tang and Trzonkowski.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Jeffrey A. Bluestone, jbluest@gmail.com
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.